Intratumoral pan-ErbB targeted CAR-T for head and neck squamous cell carcinoma: interim analysis of the T4 immunotherapy study

Background Locally advanced/recurrent head and neck squamous cell carcinoma (HNSCC) is associated with significant morbidity and mortality. To target upregulated ErbB dimer expression in this cancer, we developed an autologous CD28-based chimeric antigen receptor T-cell (CAR-T) approach named T4 immunotherapy. Patient-derived T-cells are engineered by retroviral transduction to coexpress a panErbB-specific CAR called T1E28ζ and an IL-4-responsive chimeric cytokine receptor, 4αβ, which allows IL-4-mediated enrichment of transduced cells during manufacture. These cells elicit preclinical antitumor activity against HNSCC and other carcinomas. In this trial, we used intratumoral delivery to mitigate significant clinical risk of on-target off-tumor toxicity owing to low-level ErbB expression in healthy tissues. Methods We undertook a phase 1 dose-escalation 3+3 trial of intratumoral T4 immunotherapy in HNSCC (NCT01818323). CAR T-cell batches were manufactured from 40 to 130 mL of whole blood using a 2-week semiclosed process. A single CAR T-cell treatment, formulated as a fresh product in 1–4 mL of medium, was injected into one or more target lesions. Dose of CAR T-cells was escalated in 5 cohorts from 1×107−1×109 T4+ T-cells, administered without prior lymphodepletion. Results Despite baseline lymphopenia in most enrolled subjects, the target cell dose was successfully manufactured in all cases, yielding up to 7.5 billion T-cells (67.5±11.8% transduced), without any batch failures. Treatment-related adverse events were all grade 2 or less, with no dose-limiting toxicities (Common Terminology Criteria for Adverse Events V.4.0). Frequent treatment-related adverse events were tumor swelling, pain, pyrexias, chills, and fatigue. There was no evidence of leakage of T4+ T-cells into the circulation following intratumoral delivery, and injection of radiolabeled cells demonstrated intratumoral persistence. Despite rapid progression at trial entry, stabilization of disease (Response Evaluation Criteria in Solid Tumors V.1.1) was observed in 9 of 15 subjects (60%) at 6 weeks post-CAR T-cell administration. Subsequent treatment with pembrolizumab and T-VEC oncolytic virus achieved a rapid complete clinical response in one subject, which was durable for over 3 years. Median overall survival was greater than for historical controls. Disease stabilization was associated with the administration of an immunophenotypically fitter, less exhausted, T4 CAR T-cell product. Conclusions These data demonstrate the safe intratumoral administration of T4 immunotherapy in advanced HNSCC.


Supplemental Methods
Flow cytometric monitoring of circulating T4 + CAR T-cells EDTA anticoagulated blood (50µL) was added to a FACS tube to which 4µL biotinylated anti-hEGF antibody (R&D systems, code BAF236) was added/ mixed for 15 minutes. Next, 1µL of Streptavidin-PE (ThermoFisher, code S866) was added/ mixed for 15 minutes. Then, 450µL of red blood cell lysis buffer (Biolegend 420301) was added, mixed and incubated for 15 minutes. Pre-mixed Countbright absolute counting beads (50µL; C36950, Invitrogen, Waltham, MA) were then added. Comparison was made to a positive control (T4 + T-cells) and a negative control in which primary antibody had been omitted.

MAGE-A3/A4 Interferon-γ ELISPOT Assay
Analysis was performed using a human interferon (IFN)-γ T-cell Elispot assay (U-CyTech, Utrecht, The Netherlands). Thawed peripheral blood mononuclear cells (PBMC) were resuspended in RPMI (ThermoFisher Scientific)+10% AB serum (Sigma-Aldrich, Gillingham, UK). Triplicates of 1x10 6 PBMC per well were co-cultured with peptide pools of MAGE-A3/A4 (Miltenyi Biotec, Bergisch Gladbach, Germany) at 2μg, 1µg and 0.5µg of each peptide/mL.  MAGE-A3 and MAGE-A4 ELISPOT analysis. PBMC from the indicated subjects were isolated prior to and 29 days after T4 immunotherapy. Samples were stimulated with (A) MAGE-A3 and (B) MAGE-A4 peptide pools, making comparison with unstimulated control wells. Interferon-γ spots were enumerated and data expressed as a stimulation index with respect to unstimulated control wells. Serial leukocyte counts, C-reactive protein, and ferritin post CAR T-cell immunotherapy. Neutrophil count (A), neutrophil to lymphocyte ratio (NLR; B), lymphocyte count (C), C-reactive protein (CRP; D) and ferritin (E) were measured in peripheral blood at the indicated timepoints following administration of T4 immunotherapy.

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Single Cells
Live-Dead-UV

Single Cells
Live-Dead-UV

Supplemental Figure S3
Gating strategy used to prepare Figure 4. A healthy control and representative clinical grade batch (CAR-HNC16) are shown.

Supplemental Tables
Supplemental Table S1.
Release testing of T4 immunotherapy. Labeling efficiency** Radiolabel incorporation Minimum 30% * Results of final sterility testing was not available at the time of product administration. All interim BacT/ALERT cultures and mycoplasma PCR tests had to be negative to permit product release.
** pertains to preparation of T4 radiotracer from an aliquot of the drug substance, which was undertaken in a single case.
Supplemental Table S2. Dose and volume of T4 immunotherapy per cohort.

Cohort
Target cell dose Acceptable dose range of T4 + cells
RNAScope probe for the SFG retroviral vector.